Inositol (1,4,5) Trisphosphate 3 Kinase B Controls Positive Selection of T Cells and Modulates Erk Activity

Inositol (1,4,5) trisphosphate 3 kinase B controls positive selection of T cells and modulates Erk activity

Ben G. Wen, Mathew T. Pletcher, Masaki Warashina, Sun Hui Choe, Niusha Ziaee, Tim Wiltshire, Karsten Sauer*, and Michael P. Cooke*

Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121

Edited by Peter G. Schultz, The Scripps Research Institute, La Jolla, CA, and approved January 27, 2004 (received for review October 24, 2003) The mechanisms governing positive selection of T cells in the were unable to detect expression of Itpkb in thymocytes from thymus are still incompletely understood. Here, we describe a mutant mice. N-ethyl-N-nitrosourea induced recessive mouse mutant, Ms. T-less, Itpkb, also known as inositol (1,4,5) 3 kinase B, converts which lacks T cells in the peripheral blood because of a complete inositol (1,4,5) trisphosphate (IP3) to inositol (1,3,4,5) tetrakis- ؉ ؉ block of thymocyte development at the CD4 CD8 stage. Single phosphate (IP4) (6). IP3 is a critical mediator of TCR induced nucleotide polymorphism mapping and candidate gene sequenc- Ca2ϩ release from internal stores (7). Several studies suggest ing revealed a nonsense mutation in the inositol (1,4,5) trisphos- roles for IP4 in calcium signaling in nonlymphoid cells, possibly phate 3 kinase B (Itpkb) gene in Ms. T-less mice. Accordingly, Ms. by modulating the levels of IP3 (8–10). T-less thymocytes do not show detectable expression of Itpkb Mammals express three Itpk isoforms: Itpka, Itpkb, and Itpkc protein and have drastically reduced basal inositol (1,4,5) trisphos- (6, 11, 12). Itpka and Itpkb are regulated through the binding of phate kinase activity. Itpkb converts inositol (1,4,5) trisphosphate Ca2ϩ͞calmodulin. Disruption of the brain-enriched Itpka gene to inositol (1,3,4,5) tetrakisphosphate, soluble second messengers results in minor enhancements of long-term potentiation in the 2؉ 2؉ that have been implicated in Ca signaling. Surprisingly, Ca CA1 region of the hippocampus; yet no other major defects have responses show no significant differences between wild type (WT) been noted in these mice (13). This mild phenotype may reflect and mutant thymocytes. However, extracellular signal-regulated functional redundancy with Itpkb, which shows an overlapping kinase (Erk) activation in response to suboptimal antigen receptor expression pattern (Fig. 5, which is published as supporting stimulation is attenuated in Ms. T-less thymocytes, suggesting a information on the PNAS web site). However, Itpkb is also role for Itpkb in linking T cell receptor signaling to efficient and enriched in lymphoid tissues. Itpkc shows a broader tissue sustained Erk activation. expression pattern. Surprisingly, we did not detect any significant effects on he development of mature T cells is a tightly regulated calcium responses in TCR-stimulated CD4ϩCD8ϩ T cells from Tprocess that has been studied extensively at the cellular and Ms. T-less mice. Instead, we found a specific defect in the molecular level. Lymphoid precursors destined to become T cells activation of extracellular signal-regulated kinase (Erk), a crit- arrive in the thymus from the bone marrow, where they face a ical mediator of positive selection (1). This result identifies Itpkb gauntlet of checkpoints to determine their ultimate fate (re- as a unique link between the TCR and the Ras mitogen-activated viewed in ref. 1). Briefly, T cell development in the thymus can protein kinase (MAPK) pathway, which is essential for T cell be followed by the expression of the two T cell receptor (TCR) development. coreceptors CD4 and CD8. Thymocytes at the earliest develop- Ϫ Ϫ mental stage are CD4 CD8 double negative cells. After suc- Materials and Methods ␤ cessful rearrangement of the TCR chain, they undergo rapid Mice. All mice used in this study were between 6 and 12 weeks proliferation and begin expressing both coreceptors simulta- ϩ ϩ of age. ENU mutagenized C57BL͞6 mice were generated as neously, thereby entering the CD4 CD8 double positive (DP) described (14). Mice were maintained by backcrossing affected stage. At this stage, the TCR ␣ chain is rearranged and expressed animals to C57BL͞6 and housed in the Genomics Institute of the on the cell surface to form a functional receptor. In addition, DP ϩ ϩ Novartis Research Foundation Specific Pathogen Free animal cells face a fate decision to either become mature CD4 or CD8 facility. All procedures were approved by the Genomics Institute single positive T cells or to die. This fate decision is directed by of the Novartis Research Foundation Institutional Animal Care the avidity and affinity of the TCR on DP thymocytes to self peptides presented by MHC class I or class II molecules (re- and Use Committee. viewed in ref. 2). Cells that recognize the peptide–MHC complex Flow Cytometry. with high or no affinity die by apoptosis during negative selection Single cell suspensions of thymus, lymph node, or by neglect, respectively. Cells that recognize the peptide– or spleen were stained with FITC-, phycoerythrin-, peridinin chlorophyll protein-, and allophycocyanin-conjugated antibod- MHC complex with intermediate affinity are positively selected ␤ to mature into CD4ϩ or CD8ϩ T cells. The molecular events ies against B220, TCR , CD4, CD8, CD3, CD69, CD44, CD45.1, dictating this differentiation process are an area of intense and CD45.2 (Pharmingen and eBioscience, San Diego). Cells investigation (reviewed in refs. 3 and 4). were analyzed by flow cytometry on a FACSCalibur flow In an attempt to find mediators of immune function, we are conducting a forward genetics screen in mice by using N-ethyl- This paper was submitted directly (Track II) to the PNAS office. N-nitrosourea (ENU) (5). Here, we describe Ms. T-less,a Abbreviations: ENU, N-ethyl-N-nitrosourea; DP, double positive; TCR, T cell receptor; Itpkb, recessive mouse mutant that lacks peripheral T cells because of inositol (1,4,5) trisphosphate 3 kinase B; IP3, inositol (1,4,5) trisphosphate; IP4, inositol a nearly complete block of T cell development at the DP stage. (1,3,4,5) tetrakisphosphate; Erk, extracellular signal-regulated kinase; PIP2, phosphatidyl- Single-nucleotide polymorphism mapping and candidate gene inositol (4,5) bisphosphate; GAP, GTPase-activating protein; MAPK, mitogen-activated sequencing revealed a nonsense mutation in the Itpkb gene. The protein kinase; GRP, guanine nucleotide-releasing protein. mutant allele encodes a N-terminally truncated protein, which *To whom correspondence may be addressed. E-mail: [email protected] or [email protected]. lacks the lipid kinase domain. We used various antibodies but © 2004 by The National Academy of Sciences of the USA

5604–5609 ͉ PNAS ͉ April 13, 2004 ͉ vol. 101 ͉ no. 15 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0306907101 Downloaded by guest on September 27, 2021 Fig. 1. Ms. T-less mice display a paucity of peripheral T cells and a CD4ϩCD8ϩ block in T cell development. (A) Peripheral blood lymphocytes from mutant and control mice on a C57BL͞6J background were stained with antibodies to CD3, B220, CD4, and CD8. The scatter plots show lymphocyte subpopulations as % of total lymphocytes. Thymocytes from WT (wt) and mutant (mut) mice were stained with antibodies against CD4 and CD8 to follow T cell development (B) and with antibodies to activation markers, including CD69, CD3, and TCR␤ (C). The histograms in C are gated on CD4ϩCD8ϩ thymocytes. (D) Spleens from WT and mutant mice were stained with antibodies against CD4 and CD8 to analyze the peripheral T cell compartment.

cytometer (Becton Dickinson). Acquisition and analysis were Immunoblotting and Northern Analysis. Itpkb was immunoprecipi- performed with CELLQUEST (Becton Dickinson) and FLOWJO tated from whole thymocyte extracts with an antibody against (TreeStar, Ashland, OR) software. the N-terminal region of rat Itpkb (Santa Cruz Biotechnology). Precipitate eluates or whole cell lysates from sorted DP thymo- -Analysis of Ca2؉ Responses. The protocol for measuring intracel- cytes were separated by SDS͞PAGE, transferred to nitrocellu lular calcium levels by flow cytometry was derived from L. B. lose, probed with the N-terminal antibody, and developed by Dustin (15). Thymocytes at 10 ϫ 106͞ml were labeled in DMEM enhanced chemiluminescence (ECL, Amersham Pharmacia). ϩ 10 mM Hepes with 2 ␮M Fura red (Molecular Probes), 1 ␮M For Northern blot analysis, RNA was isolated from whole Fluo-4 (Molecular Probes), and 0.2% Pluronic (Molecular thymocytes or sorted DP thymocytes, separated on a denaturing Probes) for 30 min at room temperature. Cells were washed formaldehyde-agarose gel, transferred to nylon, and hybridized twice in DMEM ϩ 10 mM Hepes with 1% FCS and rested for with a radioactive probe against an internal portion of the Itpkb 20 min in the dark. For stimulation, labeled cells were incubated transcript. with biotinylated ␣CD3 and ␣CD4 (Pharmingen and eBio- science) on ice for 15 min, washed, then resuspended with Erk Activation. Thymocytes were incubated with biotinylated prewarmed streptavidin in Hanks’ balanced salt solution (HBSS) ␣CD3 and͞or ␣CD4 for 30 min at 4°C with rotation, followed by 2ϩ with EGTA or CaCl2, and analyzed by flow cytometry. Ca stimulation with prewarmed streptavidin or phorbol 12- mobilization was determined ratiometrically as described in ref. myristate 13-acetate at the indicated time points. Stimulation 15. For single-cell calcium imaging, we adapted a protocol was stopped by adding ice-cold PBS. Cells were then washed and described in ref. 16. Briefly, thymocytes were isolated as above, lysed. Protein lysates were subjected to SDS͞PAGE, transferred labeled with 1 ␮M Fura-2 (Molecular Probes) and 0.2% Plu- to nitrocellulose, probed with antibodies to Erk1͞Erk2 or phos- ronic, stained with biotinylated ␣CD3 and ␣CD4, and adhered pho-Erk1͞Erk2 (Cell Signaling Technology), and detected by to poly(L-lysine)-treated coverslips. Cells were stimulated by enhanced chemiluminescence. perfusion of streptavidin and imaged on an inverted microscope (Nikon) under 40ϫ magnification with an UV light source. Supporting Materials and Methods. Further information can be Images were acquired over time at 340 nm and 380 nm, and the found in Supporting Materials and Methods, which is published as ͞ A340 A380 ratio was used to determine the relative intracellular supporting information on the PNAS web site. Ca2ϩ concentration. Results IP3 Kinase Activity Assay. Itpk activity in thymocyte lysates were Identification of an ENU Mouse Mutant with No Peripheral Blood T performed essentially as described in ref. 17. Briefly, 3H-inositol Cells. By using ENU mutagenesis to generate mice with defects was added to whole cell lysates. After incubation, the various in lymphoid development, we identified Ms. T-less, a mutant with inositol polyphosphates formed were resolved by thin layer a specific lack of peripheral T cells (Fig. 1A). The decrease in ϩ ϩ ϩ chromatography. Itpk activity was determined by measuring IP3 CD3 cells encompassed both CD4 and CD8 T cells. There IMMUNOLOGY conversion to IP4. were no obvious alterations in other peripheral blood cells

Wen et al. PNAS ͉ April 13, 2004 ͉ vol. 101 ͉ no. 15 ͉ 5605 Downloaded by guest on September 27, 2021 analyzed, including B220ϩ B cells. Mutant mice displayed this phenotype in a manner representative of a recessive trait and were obtained at expected Mendelian frequencies (data not shown). They showed no gross physical abnormalities or overt behavioral defects (data not shown).

Ms. T-less Mice Display a Block in T Cell Development at the CD4؉CD8؉ Stage. To determine the cause of the lack of T cells in the peripheral blood, we analyzed T cell development in the thymus. We found a nearly complete block at the CD4ϩCD8ϩ DP stage (Fig. 1B). The DP cells from mutant mice do not efficiently up-regulate activation markers, such as CD69; nor do they increase cell surface expression of CD3 or the TCR ␤ chain (Fig. 1C). The lack of CD69ϩ, CD3hi, and TCR␤hi cells in the DP population suggests that Ms. T-less thymocytes are unable to respond to or properly translate signals emanating from the TCR. Thymic cellularity of age- and sex-matched mutant mice is slightly larger than that of WT mice, suggesting that there are no gross proliferative defects in Ms. T-less thymocytes (Fig. 6, which is published as supporting information on the PNAS web site). Concurrent with a block of T cell development at the DP stage, development of ␥␦ T cells appears unaffected (data not shown). ␥␦ T cells differentiate from ␣␤ T cells before the DP stage. Therefore, the developmental block in Ms. T-less mice is specific for the ␣␤ T cell compartment, likely resulting from a specific defect in thymic selection rather than a generalized impairment of T cell development. Fig. 2. Ms. T-less thymocytes lack functional Itpkb protein. (A) Lack of The spleen and lymph nodes of Ms. T-less mice do not contain ϩ full-length Itpkb protein in Ms. T-less thymocytes. (Left) Thymocytes were significant numbers of CD4 T cells, although a small population immunoprecipitated with an antibody against Itpkb, followed by SDS͞PAGE of these cells do accumulate in older mice (Fig. 1D and data not and immunoblot analysis. (Right) Whole-cell lysates from sorted CD4ϩCD8ϩ shown). Expression of the TCR ␤ chain and high levels of CD44 cells were analyzed for the presence of Itpkb protein. All immunoblots were (data not shown) suggest that these cells may have expanded in probed with an antibody against an N-terminal peptide of Itpkb. (B) Northern a manner reminiscent of homeostatic proliferation to fill a blot analysis of Itpkb expression in sorted WT and mutant CD4ϩCD8ϩ thymo- lymphopenic environment (18, 19). cytes with an internal probe (Upper). (Lower) Expression of GAPDH as loading control. (C) Reduced IP3 kinase activity in Ms. T-less thymocytes. Extracts from WT (black bars) and mutant (white bars) thymocytes were incubated with The Defect in T Cell Development Is Inherent to the T Cells. The DP 3H-inositol for the indicated times, after which inositol polyphosphates were block of T cell development in Ms. T-less mice is reminiscent of separated by thin layer chromatography and quantiﬁed. Shown are IP4 levels the phenotype seen in mice lacking both MHC I and MHC II as the percent of total 3H-inositol converted. The rate constants of the reac- proteins (20, 21). However, we found no major differences in tion, kobs, which describe the rate of conversion of IP3 to IP4, for WT and MHC protein expression between WT and mutant animals (data mutant extracts are 0.02 minϪ1 and 0.01 minϪ1, respectively. not shown). One possibility is that an unknown ligand necessary for differentiation into mature CD4ϩ or CD8ϩ T cells is presented on the thymic epithelium and lacking in Ms. T-less mice. region did not reveal any obvious candidate genes known to be To address this issue, we performed bone marrow reconstitution involved in thymic development. Thus, we examined the expres- experiments into lethally irradiated, B6.SJL hosts. The irradia- sion status of most known or predicted genes in the region by tion depletes the hosts of their complement of hematopoietic using the Genomics Institute of the Novartis Research Founda- cells and precursors but keeps the thymic epithelium intact. We tion Gene Expression Atlas (http:͞͞expression.gnf.org) (23). We observed a profound block of T cell development at the DP stage found that the Itpkb transcript accumulates in both murine and in hosts reconstituted with Ms. T-less bone marrow (Fig. 7, which human lymphoid tissues, especially the thymus (Fig. 5 B and C). is published as supporting information on the PNAS web site). Sequencing of this candidate gene revealedaTtoAtransversion In addition, no T cells were present in the periphery, although at position 596 of the transcript, changing the codon encoding B cells reconstituted efficiently (data not shown). Bone marrow cysteine 199 to a stop (Fig. 8B). The mutant transcript encodes from WT mice exhibited normal T cell development in the host an N-terminally truncated Itpkb protein lacking most of its thymus. WT bone marrow reconstitution into lethally irradiated structure, including domains that are involved in targeting and Ms. T-less hosts exhibited normal T cell development in the regulation, as well as the catalytic domain. Immunoblot analyses thymus (data not shown). These findings indicate that the de- ϩ ϩ of lysates from sorted CD4 CD8 DP cells or of immunopre- velopmental block in T cell maturation is intrinsic to the de- cipitates from whole thymocyte extracts revealed that Ms. T-less veloping thymocytes and does not depend on ligands on, or signals from, the thymic epithelium. thymocytes lack full-length Itpkb protein (Fig. 2A). Expression of Itpkb RNA, however, is quite abundant in sorted mutant DP Ms. T-less Mice Harbor a Nonsense Mutation in Itpkb. thymocytes (Fig. 2B). In agreement with these data, extracts To determine Ϸ the genetic lesion underlying the Ms. T-less phenotype, mutant from Ms. T-less thymocytes showed an 50% reduction of Itpk mice on a C57BL͞6 background were crossed to WT 129SvJ activity compared with WT extracts (Fig. 2C). This residual mice. Single-nucleotide polymorphism genotyping of multiple activity could reflect low-level thymic expression of other Itpk phenotypically mutant or WT F2 offspring revealed a perfect isoforms (6, 11, 12, 24). Our data suggest that lack of full-length phenotype–genotype correlation for a 2-megabase interval dis- Itpkb protein expression and concomitant reduction of Itpk tal on chromosome 1 (Fig. 8A, which is published as supporting activity in thymocytes underlies the defect in T cell development information on the PNAS web site, and ref. 22). Analysis of this in Ms. T-less mice.

5606 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0306907101 Wen et al. Downloaded by guest on September 27, 2021 Fig. 3. Normal Ca2ϩ responses but impaired Erk activation after TCR stimulation in Ms. T-less thymocytes. (A) Calcium responses of bulk DP thymocyte populations from stimulated WT (green lines) or mutant (orange lines) and unstimulated WT (red lines) or mutant (blue lines) mice were analyzed by ﬂow cytometry. Cells were either stimulated with ␣CD3͞␣CD4 followed by streptavidin crosslinking (Upper) or thapsigargin (Thaps) (Lower), and responses were measured in the absence (Left) or presence (Right) of exogenous Ca2ϩ. The arrows indicate the times when streptavidin, thapsigargin, or ionomycin (Iono) were added. (B) Single-cell measurements of intracellular Ca2ϩ concentrations over time. WT and mutant thymocytes were labeled with Fura-2, and single cells were imaged after stimulation with ␣CD3͞␣CD4͞streptavidin, followed by addition of ionomycin. Relative intracellular Ca2ϩ concentrations were determined ratiometrically. (Upper) Several traces superimposed. (Lower) Traces of a single cell. (C) Thymocytes from WT and Ms. T-less mutant mice were isolated and stimulated as indicated at the indicated time points. Activated Erk1 and Erk2 were detected with an antibody speciﬁc to the phosphorylated forms of those proteins. Total Erk1 and Erk2 were also detected to determine equal loading of the samples.

-Normal Ca2؉ Responses in Ms. T-less Thymocytes. Itpkb converts IP3 modulation of histamine-induced Ca2ϩ oscillations (28). Sur to IP4. IP3 is a well characterized second messenger involved in prisingly, single-cell imaging of Fura-2 labeled thymocytes did calcium signaling (25). TCR ligation leads to activation of not reveal significant differences in magnitude or periodicity of phospholipase C␥, which hydrolyzes phosphatidylinositol (4,5) TCR-induced Ca2ϩ oscillations between WT and mutant mice bisphosphate (PIP2) to diacylglycerol and IP3. The augmenta- (Fig. 3B). Taken together, these data suggest that defects in Ca2ϩ tion of intracellular IP3 levels triggers the release of Ca2ϩ from signaling are unlikely to underlie the profound defect in T cell internal stores by means of IP3 receptors (26). In Jurkat cells, development observed in Ms. T-less mice. Itpk activity and IP4 production are elevated during TCR stimulation (27). Thus, Itpkb could serve to limit TCR-induced Defective Erk Activation in Ms. T-less Thymocytes: A Role for Itpkb in Ca2ϩ mobilization through conversion of IP3 to IP4. However, Ras Signaling. The lack of an overt effect on Ca2ϩ responses in Ms. it has also been postulated that IP4 can potentiate IP3 signaling T-less thymocytes led us to consider other mechanisms of how through the specific inhibition of a 5Ј-phosphatase that hydro- Itpkb could control T cell selection. Because IP3 levels do not lyzes IP3 to inositol (1,4) bisphosphate (10). Thus, formation of seem to be affected in these mice (unpublished observations), we IP4 could also affect Ca2ϩ mobilization positively. We therefore addressed putative mechanisms involving IP4. The protein GT- investigated whether defects in Ca2ϩ signaling might underlie the Pase-activating protein (GAP)1IP4BP has been shown to bind IP4 developmental defect in Ms. T-less thymocytes. with high affinity and specificity in vitro (29). GAP1IP4BP is a As shown in Fig. 3A, bulk Ca2ϩ responses to stimulation protein that stimulates the small GTPase Ras to convert GTP to through the TCR or with thapsigargin, a chemical that depletes GDP, rendering Ras inactive. Several studies have demonstrated internal Ca2ϩ stores and bypasses the requirement for IP3 essential roles for the Ras pathway in T cell development. generation, are similar between WT and mutant thymocytes in Functional inactivation of the Ras activator Ras guanine nucle- the absence or presence of external Ca2ϩ. Thus, Ms. T-less otide-releasing protein (RasGRP) (30), Ras (31), or components thymocytes have no major defects in internal Ca2ϩ release or of the MAPK pathway downstream of Ras (32–35) all affect external Ca2ϩ influx. We next investigated the Ca2ϩ responses of maturation of DP thymocytes and positive selection. Therefore, individual cells to TCR stimulation. Thymocytes undergoing GAP1IP4BP could be an important component that connects positive selection display dramatic oscillations of intracellular Itpkb-mediated IP4 production to Ras activation in T cell Ca2ϩ levels that have a periodicity on the order of seconds (16). development. Cells that do not receive signals for positive selection do not show To address activation of the Ras pathway in Ms. T-less

the same magnitude of oscillations at the single-cell level. In thymocytes, cells were stimulated with either a suboptimal TCR IMMUNOLOGY HeLa cells, Itpk activity and IP4 have been implicated in the signal by using ␣CD3 alone or with a maximal TCR signal by

Wen et al. PNAS ͉ April 13, 2004 ͉ vol. 101 ͉ no. 15 ͉ 5607 Downloaded by guest on September 27, 2021 Fig. 4. A model for the role of Itpkb and its product IP4 in the regulation of Ras signaling to Erk in thymocytes. For details, see Discussion.

using a combination of ␣CD3 and ␣CD4 antibodies (Fig. 3C). Based on our finding of impaired TCR-induced Erk activation Ras activation leads to the activation and phosphorylation of the in Ms. T-less thymocytes, we propose a model of Ras activation MAPKs Erk1 and Erk2. By using immunoblot analysis, we found that is regulated by Itpkb through the production of IP4 (Fig. 4). a significant impairment of Erk1 and Erk2 activation in response In naı¨ve thymocytes, Ras is kept in the inactive GDP-bound state to suboptimal (␣CD3 alone) stimulation in Ms. T-less thymo- by the action of RasGAPs, in this case GAP1IP4BP, which cytes. Optimal stimulation conditions (␣CD3 and ␣CD4) or hydrolyzes Ras-GTP to Ras-GDP. GAP1IP4BP is membrane- stimulation with the diacylglycerol analog phorbol 12-myristate associated through an interaction of its pleckstrin homology 13-acetate, which can localize RasGRP1 to the plasma mem- (PH) domain with membrane bound PIP2. This same PH domain brane to allow for Ras activation (36, 37), elicited normal levels can also bind water-soluble IP4; thus, the balance between of Erk1 and Erk2 activation. These data demonstrate that Ms. membrane-bound PIP2 and soluble IP4 may determine the IP4BP T-less thymocytes are unable to efficiently activate Erk1 and localization of GAP1 (29, 40). High-level expression of IP4BP Erk2 under moderate stimulation conditions. Thus, the block GAP1 is restricted to lymphoid subsets (Fig. 9, which is during positive selection in Ms. T-less thymocytes may reflect published as supporting information on the PNAS web site). In thymocytes undergoing selection, TCR stimulation activates critical roles for Itpkb and its product IP4 as regulators of ␥ TCR-induced Ras activation. phospholipase C , which hydrolizes PIP2 to diacylglycerol and IP3. Diacylglycerol recruits the Ras-activating RasGRP1 to the Discussion plasma membrane, which activates Ras through an exchange of By using ENU mutagenesis, we have identified Ms. T-less,a GDP for GTP and initiates the MAPK cascade, leading to Erk mouse mutant with a paucity of peripheral T cells resulting from activation (41). At the same time, hydrolysis of PIP2 weakens the ϩ ϩ interaction of GAP1IP4BP with the plasma membrane. Accumu- a block of ␣␤ T cell development at the CD4 CD8 DP stage ϩ lation of IP3 triggers Ca2 release from intracellular stores and with a phenotype suggestive of impaired positive selection. This ϩ ϩ subsequent Ca2 influx, allowing for Ca2 -dependent activation defect is intrinsic to the developing T cells and can be attributed ϩ ϩ of Itpkb, possibly through Ca2 ͞calmodulin and Ca2 ͞calmod- to a loss-of-function mutation in the gene Itpkb. Although Itpkb ulin-dependent kinase II (42, 43). Itpkb converts IP3 to IP4, converts IP3, an important second messenger that mediates which may sequester GAP1IP4BP in the cytoplasm by competition Ca2ϩ responses to TCR stimulation, into IP4, which has been 2ϩ with the already low levels of PIP2 for binding to its PH domain. implicated in modulating Ca oscillations in HeLa cells, we IP4BP 2ϩ Sequestration of GAP1 in the cytoplasm allows unhindered found no obvious changes in IP3 levels or Ca responses in Ras activation at the plasma membrane. In Ms. T-less mutant thymo- mutant thymocytes stimulated by the TCR. These findings cytes that lack Itpkb, a severe reduction in the generation of IP4 could corroborate previous reports that nonspecific Itpk inhibition in presumably prevent the efficient removal of GAP1IP4BP from the 2ϩ Jurkat cells with adriamycin did not affect TCR-induced Ca plasma membrane and thereby prevent sufficient Ras and Erk activa- mobilization (38). We have, however, identified a significant tion to allow for positive T cell selection. defect in Erk activation after suboptimal stimulation of Ms. T-less Our model provides an intriguing rationale for the defects in thymocytes, which may be a direct consequence of insufficient TCR-induced Erk activation and positive selection of Ms. T-less Ras activation. thymocytes caused by impaired Itpkb function. However, recent Our data substantiate and suggest a mechanistic explanation data suggest that the regulation of Ras activation may be more for the similar T cell developmental phenotype resulting from complex. First, T cells express another RasGAP, Ca2ϩ-promoted disruption of the Itpkb gene, which was reported by Pouillon et Ras inactivation (CAPRI), in addition to GAP1IP4BP (44). al. (39) while this manuscript was in preparation. The authors Second, a recent study suggests that in various cell types, ϩ found similar defects in positive selection and unimpaired Ca2 including Jurkat cells, Ras exists both at the plasma membrane signaling in mice lacking Itpkb, even on transgenic TCR back- and on the surface of the Golgi compartment (45). However, grounds. only Golgi-associated Ras is activated in response to TCR

5608 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0306907101 Wen et al. Downloaded by guest on September 27, 2021 stimulation by a mechanism involving RasGRP recruitment to Erk-MAPK signaling pathway (32–34) or chemical modulation the Golgi membrane in a phospholipase C␥-dependent manner. of the Ras-Erk pathway (47, 48) can affect positive selection of In contrast, plasma membrane Ras is kept inactive through a T cells and alter lineage commitment. Collectively, these studies Ca2ϩ-mediated, PIP2͞PIP3-independent plasma membrane re- suggest that the amount of Ras activation is important for the cruitment of CAPRI. Thus, although both GAP1IP4BP and developmental decisions made by T cells undergoing selection. CAPRI are likely negative regulators of Ras activation in T cells, In this regard, Itpkb and its product IP4 may serve to translate their respective mechanisms of recruitment to membrane-bound Ras low-level TCR signaling that occurs during positive selection into differ, and they are likely to be differentially affected by IP4. It will be levels of Ras-Erk activation that are sufficient to allow for interesting to investigate whether GAP1IP4BP controls activation of the thymocyte maturation. possibly more relevant Golgi-bound Ras in T cells. IP4 is a precursor for several higher order inositol polyphos- It is not surprising that Ms. T-less mutant thymocytes exhibit phates, some of which are generated in TCR-stimulated T cells a block in Erk activation only in response to suboptimal stimu- (49, 50). Their physiological roles are largely undefined (9), but lation. Most genetic studies involving the Ras pathway have recent studies in yeast suggest functions in regulating chromatin documented a dependence of T cell selection on the strength of remodeling (51, 52). Although we have no evidence for an the TCR signal, which modulates the level of Ras activation. involvement of IP4 in chromatin remodeling in thymocytes, RasGRP1Ϫ/Ϫ mice exhibit a nearly complete block in positive further investigation is required. At present, it is more intriguing selection (30), but in the context of a TCR transgene capable of to focus on the relative roles of IP4 and RasGAPs in regulating providing sufficient signal strength, positive selection can occur T cell signaling through Ras. Regardless of the precise mecha- normally (46). Additionally, transgenic mice expressing a dom- nism, this study clearly defines a critical role for Itpkb in the inant negative form of Ras (31) display impaired positive selec- regulation of thymocyte maturation. tion, a phenotype that is exacerbated when crossed to a transgenic mouse bearing a dominant negative form of MEK1, the We thank Michael Christensen, John Hogenesch, Lisa Tarantino, Mark Sandberg, John Walker, and Nathanael Gray for many helpful dis- MAPK kinase downstream of Ras activation (35). The additive cussions; our animal, genomics, sequencing, gene profiling, and bio- effect of the two transgenes again points to the importance of the informatics core groups for their support; and Mike Young, Carie magnitude of Ras activation in the differentiation of immature Dubord-Jackson, and Chris Trussel for their expert help with all thymocytes. Finally, genetic alteration of components of the fluorescence-activated cell sorter analyses.

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